Compositions and methods for targeted inactivation of HIV cell surface receptors

Abstract

Compositions for targeted mutagenesis of cell surface receptors for HIV and methods of their use are provided herein. The compositions include triplex-forming molecules that bind to duplex DNA in a sequence specific manner at target sites to form triple-stranded structures. The triplex-forming molecules can be triplex-forming oligonucleotides (TFOs) or peptide nucleic acids (PNAs). The triplex-forming molecules are useful to induce site-specific homologous recombination in mammalian cells when used in combination with donor oligonucleotides. The triplex-forming molecules target sites within or adjacent to genes that encodes cell surface receptors for human immunodeficiency virus (HIV). This binding stimulates homologous recombination of a donor oligonucleotide to cause mutations in HIV cell surface receptor genes that result in one or more deficiencies in the ability of the encoded receptor to bind to HIV and allow its transport into the cell. Methods for ex vivo and in vivo prophylaxis and therapy of HIV infection using the disclosed compositions are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Ser. No. 60 / 880,232 entitled “Targeted inactivation of the CCR5 gene using PNAs as an anti-HIV therapy”, filed Jan. 11, 2007.GOVERNMENT SUPPORT[0002]This invention was made with government support awarded by the National Institutes of Health under Grant Number NIH R01CA64186. The United States government has certain rights in this invention.FIELD OF THE INVENTION[0003]The present disclosure generally relates to the field of compositions that bind to DNA encoding cell surface receptors for HIV and methods of using these compositions.BACKGROUND OF THE INVENTION[0004]HIV-1 is a member of the Retroviridae family belonging to the genus lentiviruses. The Retroviridae are enveloped viruses containing two positive sense RNA strands that are converted into dsDNA by the highly error-prone viral reverse transcriptase enzyme generating isolate diversity by both point mutation and intergenomic recombination. HI...

AI Technical Summary

Benefits of technology

[0022]The target site is within or adjacent to a gene that encodes a cell surface receptor for human immunodeficiency virus (HIV). The HIV cell surface receptor can be a chemokine receptor, including CXCR4, CCR5, CCR2b, CCR3 and CCR1. The target site can be within the coding region of the gene. The target sequence is preferably within or adjacent to a portion of the HIV cell surface receptor gene that important to its function in allowing HIV entry into cells, such as nucleotides or nucleotide sequences involved in efficient expression of the receptor, transport of the receptor to the cell surface, stability of the receptor, viral binding by the receptor, or endocytosis of the receptor. In one embodiment, the target site for the triplex-forming molecule is within or adjacent to the human CCR5 gene. In a preferred embodiment, the target site encompasses or is adjacent to the site of a naturally occurring nonsense mutation referred to as the Δ32 mutation.

[0023]The triplex-forming molecules can be triplex-forming oligonucleotides (TFOs). TFOs are single-stranded oligonucleotides between about 7 and about 40 nucleotides in length. TFOs bind to target sites containing polypurine, homopurine, polypyrimidine or homopyrimidine base compositions within a major grove of duplex DNA. TFOs can contain chemical modifications to their nucleotide constituents, including chemical modifications of their heterocyclic bases, sugar moieties or phosphate moieties. These modifications can increase the biding affinity of the TFO for the target site or the stability of the fowled triplex.

[0024]The triplex-forming molecules can also be peptide nucleic acids (PNAs). Highly stable PNA:DNA:PNA triplex structures can be formed from strand invasion of a duplex DNA with two PNA strands. The two PNA strands can be linked together to form a bis-PNA molecule. PNAs also bind to target sites with polypurine or homopurine sequences, but can do so at shorter target sequences relative to TFOs, and with greater stability.

Problems solved by technology

A number of pharmaceutical companies are currently trying to develop entry-inhibitor drugs to block the receptor protein, although progress has been hindered by toxicity, efficacy and drug resistance.

However, while facile methods exist to introduce new genes into mammalian cells, the frequency of homologous integration is limited (Hanson et al., (1995) Mol. Cell. Biol. 15(1), 45-51), and isolation of cells with site-specific gene insertion typically requires a selection procedure (Capecchi, M. R., (1989) Science 244(4910), 1288-1292).

This approach requires complex delivery systems to introduce the replacement gene into the cell, such as genetically engineered viruses, or viral vectors.

However, in vivo efficiency is low due to the limited number of recombination events actually resulting in replacement of the defective gene.

Images